Stimulation of human extravillous trophoblast migration by IGF-II is mediated by IGF type 2 receptor involving inhibitory G protein(s) and phosphorylation of MAPK

We have earlier shown that migration and invasiveness of first trimester hu man extravillous trophoblast cells are stimulated by IGF-II, independently of IGF type 1 receptor and that migration stimulation is the primary reason for increased extravillous trophoblast cell invasiveness induced by IGF-IL . In the present study we examined the functional role of IGF type II recep tor in IGF-II stimulation of extravillous trophoblast cell migration and th e underlying signal transduction pathways including the participation of in hibitory G protein(s) and MAPK. The migratory ability of a well characteriz ed in vitro propagated human first trimester extravillous trophoblast cell line expressing the phenotype of extravillous trophoblast cells in situ was quantitated with a Transwell migration assay under different experimental conditions. We found that the extravillous trophoblast cells expressed an a bundance of IGF type 2 receptor as detected by immunostaining and Western b lots, and recombinant human IGF-II promoted their migration in a dose- and time-dependant manner. Both polyclonal and monoclonal IGF type 2 receptor-b locking antibodies blocked migration-stimulating effects of IGF-II. Two syn thetic IGF-II analogs ([Leu(27)]IGF-II, which can bind to IGF type 2 recept or and IGF-binding proteins, but not IGF type I receptor, and [QAYL-Leu(27) ]IGF-II, which can bind to IGFR-II, but neither IGFR-I nor IGF-binding prot eins) both stimulated extravillous trophoblast cell migration to levels hig her than those induced by wild-type IGF-II. These results reveal that IGF-I I action was mediated by IGF type 2 receptor, independently of IGF type I r eceptor and IGF-binding proteins. Treatment of extravillous trophoblast cel l membrane preparations with IGF-II decreased adenylyl cyclase activity in a concentration-dependant manner, indicating the participation of inhibitor y G proteins in IGF-II action. This was substantiated further with the find ings that increasing intracellular cAMP using forskolin or (Bu)(2)cAMP inhi bited basal extravillous trophoblast cell migration and blocked IGF-Il stim ulation of migration. IGF-II treatment rapidly stimulated phosphorylation o f MAPK (ERK-1 and -2), which was blocked by pretreatment of extravillous tr ophoblast cells with the ALA,PK kinase (MEK) inhibitor PD98059. Treatment w ith this inhibitor also blocked extravillous trophoblast cell migration in the presence or absence of IGF-II. These results, taken together, reveal th at IGF-II stimulates extravillous trophoblast cell migration by signaling t hrough IGF type 2 receptor, involving inhibitory G proteins and activating the MAPK pathway.